Dual bronchodilatory and pulmonary anti-inflammatory activity of RO5024118, a novel agonist at vasoactive intestinal peptide VPAC2 receptors.

BACKGROUND AND PURPOSE: Vasoactive intestinal peptide is expressed in the respiratory tract and induces its effects via its receptors, VPAC(1) and VPAC(2). RO5024118 is a selective VPAC(2) receptor agonist derived via chemical modification of an earlier VPAC(2) agonist, RO0251553. In the present studies, we characterized the pharmacological activity of RO5024118. EXPERIMENTAL APPROACH: Stability of RO5024118 to human neutrophil elastase was assessed. Bronchodilatory activity of RO5024118 was investigated in guinea pig and human isolated airway smooth muscle preparations and in a guinea pig bronchoconstriction model. Pulmonary anti-inflammatory activity of RO5024118 was investigated in a lipopolysaccharide mouse model and in a porcine pancreatic elastase (PPE) rat model. KEY RESULTS: RO5024118 demonstrated increased stability to neutrophil elastase compared with RO0251553. In human and guinea pig isolated airway preparations, RO5024118 induced bronchodilatory effects comparable with RO0251553 and the long-acting ß-agonist salmeterol and was significantly more potent than native vasoactive intestinal peptide and the short-acting ß-agonist salbutamol. In 5-HT-induced bronchoconstriction in guinea pigs, RO5024118 exhibited inhibitory activity with similar efficacy as, and longer duration than, RO0251553. In a lipopolysaccharide-mouse model, RO5024118 inhibited neutrophil and CD8(+) cells and myeloperoxidase levels. In rats, intratracheal instillation of PPE induced airway neutrophilia that was resistant to dexamethasone. Pretreatment with RO5024118 significantly inhibited PPE-induced neutrophil accumulation. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that RO5024118 induces dual bronchodilatory and pulmonary anti-inflammatory activity and may be beneficial in treating airway obstructive and inflammatory diseases. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Peptide and peptide mimetic inhibitors of antigen presentation by HLA-DR class II MHC molecules. Design, structure-activity relationships, and X-ray crystal structures.

Molecular features of ligand binding to MHC class II HLA-DR molecules have been elucidated through a combination of peptide structure-activity studies and structure-based drug design, resulting in analogues with nanomolar affinity in binding assays. Stabilization of lead compounds against cathepsin B cleavage by N-methylation of noncritical backbone NH groups or by dipeptide mimetic substitutions has generated analogues that compete effectively against protein antigens in cellular assays, resulting in inhibition of T-cell proliferation. Crystal structures of four ternary complexes of different peptide mimetics with the rheumatoid arthritis-linked MHC DRB10401 and the bacterial superantigen SEB have been obtained. Peptide-sugar hybrids have also been identified using a structure-based design approach in which the sugar residue replaces a dipeptide. These studies illustrate the complementary roles played by phage display library methods, peptide analogue SAR, peptide mimetics substitutions, and structure-based drug design in the discovery of inhibitors of antigen presentation by MHC class II HLA-DR molecules.

Peptidomimetic compounds that inhibit antigen presentation by autoimmune disease-associated class II major histocompatibility molecules.

We have identified a heptapeptide with high affinity to rheumatoid arthritis-associated class II major histocompatibility (MHC) molecules. Using a model of its interaction with the class II binding site, a variety of mimetic substitutions were introduced into the peptide. Several unnatural amino acids and dipeptide mimetics were found to be appropriate substituents and could be combined into compounds with binding affinities comparable to that of the original peptide. Compounds were designed that were several hundred-fold to more than a thousand-fold more potent than the original peptide in inhibiting T-cell responses to processed protein antigens presented by the target MHC molecules. Peptidomimetic compounds of this type could find therapeutic use as MHC-selective antagonists of antigen presentation in the treatment of autoimmune diseases.

Binding affinity independent contribution of peptide length to the stability of peptide-HLA-DR complexes in live antigen presenting cells.

The effect of peptide length on the stability of peptide-HLR-DR1 (DR1) complexes was analyzed using two peptide series of increasing length, each containing a 7mer core with five DR1-binding anchors, extended stepwise with Ala residues at the N- and C-terminus, respectively. The Ala extensions, although did not affect binding affinity, significantly increased the half lives of peptide-DR1 complexes (from 1.5 h up to 10 h) in live antigen presenting cells (APC). Flanking residues from position -2 to 0 and 8 to 11 were involved in the affinity-independent increase of complex stability. The shortest (8mer and 9mer) peptides, with in vivo half lives of <2.5 h, were unable to form stable complexes with DR1 in presence of HLA-DM (DM) molecules, and were poor competitors of antigen presentation. Longer peptides were resistant to DM-mediated unloading, and were efficient competitors of antigen presentation. Thus, DM appears to limit short peptides in establishing biologically relevant DR occupancy, despite their high binding affinity. In APC, stable complexes can form only with high affinity peptides of >9 residues, and the longevity of complexes seems to depend on full of occupation of the binding site.

Peptide dependence of major histocompatibility complex class II specific alloreactive responses.

Splenic cells from transgenic mice, in which a single peptide is complexed to all major histocompatibility complex (MHC) class II molecules, are found to be incapable of triggering primary allogeneic mixed lymphocyte/leucocyte reactions (MLR) when co-cultured with lymphocytes from MHC class II congenic mouse strains. In addition, a single HLA-DR-blocking peptide can completely abrogate the capacity of splenocytes from chimeric HLA-DR/H2-E transgenic mice to stimulate primary MLR of T cells from wild-type mice. These results indicate that the primary alloreactive response is directed against a multitude of peptides presented by allogeneic MHC molecules.

Novel cyclic peptide agonist of high potency and selectivity for the type II vasoactive intestinal peptide receptor.

Ro 25-1392 [Ac-Glu8,OCH3-Tyr10,Lys12,Nle17,Ala19,A sp25,Leu26,-Lys27,28-vasoactive intestinal peptide(cyclo 21-25)] is a cyclic peptide analog of vasoactive intestinal peptide (VIP) that potently exerts cellular effects typical of VIP. The selectivity of Ro 25-1392 for type I (VIPR1) and type II (VIPR2) VIP receptors was investigated first in competitive binding studies using Chinese hamster ovary cell transfectants stably expressing recombinant human VIPR1 and VIPR2. Nonradioactive Ro 25-1392 was as potent a competitive inhibitor as VIP for the binding of 125I-VIP to VIPR2 transfectants (Ki = 9.6 +/- 1.0 and 16 +/- 1.7 nM, respectively; mean +/- S.E.M., n = 4). In contrast, Ro 25-1392 had a very low affinity for VIPR1, compared with VIP, and attained a maximum of only 40% mean inhibition of binding of 125I-VIP at 1 microM. The affinity of VIP (Ki = 3.4 +/- 1.5 nM, mean +/- S.E.M., n = 4) for binding to VIPR1 was 1000-fold greater than that of Ro 25-1392. Ro 25-1392 evoked concurrent and concentration-dependent increases in intracellular levels of calcium and cyclic AMP (EC50 = 3.0 +/- 0.4 nM, mean +/- S.E.M., n = 4) in VIPR2 transfectants, but not in VIPR1 transfectants. The VIP receptor specificity of Ro 25-1392 was confirmed by preincubation of Chinese hamster ovary transfectants with 0.1 microM Ro 25-1392 for 18 hr at 37 degrees C, to down-regulate each type of VIP receptor. Pretreatment of VIPR2 transfectants with Ro 25-1392 decreased Bmax by a mean of 58% and VIP-induced increases in the intracellular concentration of cyclic AMP by a mean of 65%. In contrast, there was no significant change in VIPR1 transfectants after pretreatment with Ro 25-1392. Ro 25-1392 thus is selectively recognized by VIPR2, with consequent initiation of cyclic AMP and Ca+2 signals and down-regulation of VIPR2. This potent analog of VIP may prove useful for investigations of VIPR2-mediated physiological effects of VIP and exploration of the roles of VIPR2 in diseases.

HLA-DR4-IE chimeric class II transgenic, murine class II-deficient mice are susceptible to experimental allergic encephalomyelitis.

To investigate the development of HLA-DR-associated autoimmune diseases, we generated transgenic (Tg) mice with HLA-DRA-IE alpha and HLA-DRB1*0401-IE beta chimeric genes. The transgene-encoded proteins consisted of antigen-binding domains from HLA-DRA and HLA-DRB1*0401 molecules and the remaining domains from the IE(d)-alpha and IE(d)-beta chains. The chimeric molecules showed the same antigen-binding specificity as HLA-DRB1*0401 molecules, and were functional in presenting antigens to T cells. The Tg mice were backcrossed to MHC class II-deficient (IA beta-, IE alpha-) mice to eliminate any effect of endogenous MHC class II genes on the development of autoimmune diseases. As expected, IA alpha beta or IE alpha beta molecules were not expressed in Tg mice. Moreover, cell-surface expression of endogenous IE beta associated with HLA-DRA-IE alpha was not detectable in several Tg mouse lines by flow cytometric analysis. The HLA-DRA-IE alpha/HLA-DRB1*0401-IE beta molecules rescued the development of CD4+ T cells in MHC class II-deficient mice, but T cells expressing V beta 5, V beta 11, and V beta 12 were specifically deleted. Tg mice were immunized with peptides, myelin basic protein (MBP) 87-106 and proteolipid protein (PLP) 175-192, that are considered to be immunodominant epitopes in HLA-DR4 individuals. PLP175-192 provoked a strong proliferative response of lymph node T cells from Tg mice, and caused inflammatory lesions in white matter of the CNS and symptoms of experimental allergic encephalomyelitis (EAE). Immunization with MBP87-106 elicited a very weak proliferative T cell response and caused mild EAE. Non-Tg mice immunized with either PLP175-192 or MBP87-106 did not develop EAE. These results demonstrated that a human MHC class II binding site alone can confer susceptibility to an experimentally induced murine autoimmune disease.

Comparison of cyclic and linear analogs of vasoactive intestinal peptide.

A series of i-->i + 4 side-chain to side-chain lactam analogs of vasoactive intestinal peptide has been prepared in order to study the effect of cyclization on biological activity. In vitro, on guinea pig tracheal smooth muscle and on human bronchial tissue, approximately half of the cyclic analogs showed increased potency and half were decreased over the linear analogs. Several cyclic compounds were between 10- and 20-fold more potent and one was 290-fold more potent than the linear species. In vivo, in guinea pigs, the cyclic compounds showed increased potency by up to 70-fold and significantly enhanced duration of action as compared to linear compounds.

Down-regulation of class II major histocompatibility complex molecules on antigen-presenting cells by antibody fragments.

Certain HLA class II-specific monoclonal antibodies (mAb) cause up to 90% decrease in the cell surface expression of class II molecules. This down-regulation is isotype-specific, i.e. DR-specific mAb do not affect the expression of DP and DQ molecules. However, antibodies binding to one DR allotype down-regulate both allotypes in heterozygous antigen-presenting cells (APC), indicating that the phenomenon is not a direct consequence of ligation. All down-regulating mAb identified recognize the first (peptide binding) domains of class II heterodimers, and strongly inhibit the activation of class II-restricted human T cells in vitro. Conversely, non-down-regulating mAb fail to inhibit T cell activation, and most of them (four out of five) recognize class II second domains. Down-regulating antibodies are cytotoxic for B lymphoblastoid cell lines and for a small proportion of normal activated B cells. Their F(ab')2 fragments mediate both down-regulation and cytotoxicity, whereas the monovalent Fab fragments are not cytotoxic, but retain the down-regulatory and T cell inhibitory properties. These findings raise the possibility of a class II major histocompatibility complex-specific, antibody-based immunosuppressive therapy without cytotoxic side effects.

Structure-activity studies on the vasoactive intestinal peptide pharmacophore. 1. Analogs of tyrosine.

From previous work, the primary functional groups, i.e. side chains, of the vasoactive intestinal peptide which are responsible for interaction with the VIP receptor have been identified. One of these sites, the side chain of tyrosine22 is essential for high receptor affinity. The present work aims to examine this site in greater detail. Several Boc-substituted-phenylalanine derivatives were prepared and incorporated into VIP analogs as replacement for tyrosine22. These analogs, of the form Ac-[Lys12,Nle17,X22,Val26,Thr28]-VIP, were assayed as smooth muscle relaxants and found to be full agonists of native VIP. Most of the analogs, however, proved to be less potent than the parent analog by up to 300-fold. A few analogs, all possessing electron-donating substituents, retained nearly full potency. Two compounds, 3-F,4-OH-Phe, 42 and 3-OCH3,4-OH-Phe, 43, were found to be 1.5- and 3.4-fold more potent than the parent compound, which equates to being 8.9- and 20-fold more potent than native VIP. Compound 43 was also found to be active as a bronchodilator in vivo in guinea pigs, with slightly over 2-fold enhanced potency and a significantly longer duration of action (> 20 min) when compared to the parent compound (5 min). The physical characteristics of the various substituents and their effect on biological activity are discussed with a brief analysis by QSAR techniques.

Down-regulation of class II major histocompatibility complex molecules on antigen presenting cells after interaction with helper T cells.

The recognition of antigenic peptides by CD4+ helper T cells is demonstrated here to result in a dramatic (up to 90%) decrease in expression of major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells (APC). The reduction is selective to the class II isotype presenting the antigen, but if affects both allelic forms of the same isotype in heterozygous APC. The observed MHC down-regulation requires a specific T cell receptor-peptide-class II interaction, a direct contact between T cell and APC, and the involvement of CD2 molecules. These findings have important implications for the regulation of immune response, self tolerance, and autoimmunity.

Design and development of a vasoactive intestinal peptide analog as a novel therapeutic for bronchial asthma.

Analogs of vasoactive intestinal peptide (VIP) were synthesized and screened as bronchodilators with the ultimate goal of enhancing the potency and extending the duration of action of the native peptide. Several design approaches were applied to the problem. First, the amino acid residues required for receptor binding and activation were identified. A model of the active pharmacophore was developed. With knowledge of the secondary structure (NMR) of the peptide, various analogs were synthesized to stabilize alpha-helical conformations. Having achieved a level of enhanced bronchodilator potency, our approach then concentrated on identification of the sites of proteolytic degradation and synthesis of metabolically-stable analogs. Two primary cleavage sites on the VIP molecule were identified as the amide bonds between Ser25-Ile26 and Thr7-Asp8. This information was used to synthesize cyclic peptides which incorporated disulfide and lactam ring structures. Analog work combined the best multiple-substitution sites with potent cyclic compounds which resulted in identification of a cyclic lead peptide. This compound, Ro 25-1553, exhibited exceptionally high potency, metabolic stability, and a long duration of action and may be an effective therapeutic for the treatment of bronchospastic diseases.

Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part II: Effect on in vitro and in vivo models of pulmonary anaphylaxis.

Studies were conducted to compare the effect of native vasoactive intestinal peptide (VIP), Ro 25-1553 (a cyclic peptide analog of VIP) and salbutamol (a beta2-adrenoceptor agonist) on antigen-induced pathophysiological effects in the guinea pig. Ro 25-1553 and salbutamol (0.01-1.0 microM) prevented antigen-induced contractions of the guinea pig trachea in vitro with IC50 values of 0.07 and 0.05 microM, respectively. VIP (0.01-1.0 microM) had no effect on antigen-induced tracheal contractions. Aerosolized Ro 25-1553 and salbutamol were equipotent in preventing antigen-induced increases in guinea pig lung resistance (IC50 value = 0.0001%), whereas aerosolized VIP (0.1%) was ineffective. Ro 25-1553 (0.1-100 micrograms), instilled intratracheally 2 min before the antigen challenge of buffer-perfused lungs from sensitized guinea pigs, produced a dose-dependent inhibition of bronchoconstrictor, vasoconstrictor and edemagenic responses, whereas intratracheal VIP (100 micrograms) had no effect. Intratracheal salbutamol (0.1-100 micrograms) inhibited antigen-induced responses in a manner comparable to Ro 25-1553. Lung inflammation was assessed as leukocyte accumulation in bronchoalveolar lavage fluid after the antigen provocation. Aerosolized antigen-induced bronchoalveolar lavage eosinophilia (13-fold increase over saline controls) at 6 hr after challenge was prevented in a concentration-dependent manner by pretreatment with nebulized Ro 25-1553 and salbutamol, but not by pretreatment with native VIP. These results indicate that Ro 25-1553 suppresses various pathophysiological features associated with pulmonary anaphylaxis and asthma, including airway reactivity, edema formation and granulocyte accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part I: In vitro and in vivo bronchodilator studies.

Ro 25-1553, a cyclic peptide analog of vasoactive intestinal peptide (VIP), was designed to overcome many of the deficiencies inherent in this natural neuropeptide. On isolated guinea pig tracheal smooth muscle, Ro 25-1553 produces concentration-dependent relaxation of contractile responses to a number of different spasmogens. Depending on the contractile stimulus, Ro 25-1553 is 24 to 89 times more potent than VIP as a relaxant of guinea pig trachea. The high potency of Ro 25-1553 extends to studies on isolated, histamine-contracted, human bronchial smooth muscle, where Ro 25-1553 exhibits a 390-fold enhancement over native VIP and is more potent than other bronchodilating drugs, such as the beta 2-adrenoceptor agonists isoproterenol and salbutamol. Ro 25-1553 was shown to displace the radioligand 125I-VIP from rat forebrain membranes with an IC50 value of 4.98 nM, thereby demonstrating that it acts at a VIP receptor. In addition, when tested in a battery of 40 other binding assays (e.g., muscarinic, histamine, LTs, Ca++, TxA2, endothelin, alpha and beta adrenergic, platelet-activating factor, neurokinins, etc.) at concentrations as high as 10 microM, Ro 25-1553 was found to be inactive; thus it appears to be specific for VIP receptors. The potent smooth muscle relaxant activity exhibited in vitro by Ro 25-1553 is also evident after in vivo intratracheal administration or aerosolization of the compound. Pulmonary responses evoked by histamine, leukotriene D4, platelet-activating factor and acetylcholine are inhibited dose-dependently by intratracheally instilled Ro 25-1553 with nearly identical potency (ED50 values ranging from 0.07 micrograms to 0.26 micrograms).(ABSTRACT TRUNCATED AT 250 WORDS)

Vasoactive intestinal peptide: role of calmodulin and catalytic antibodies.

In the accompanying article Paul and Ebadi [1993 (Neurochem Int. 23, 197-214)] review recent observations that the neuropeptide vasoactive intestinal peptide (VIP) and the related peptide growth hormone releasing factor (GRF) bind to two novel protein molecules unrelated to their G-protein coupled receptors. VIP and GRF have been reported to bind with high affinity to calmodulin, leading to modulation of biological activities of the regulatory protein. VIP has also been reported to bind to autoantibodies isolated from both normal and asthmatic subjects. Several of these autoantibodies have been found to catalyze the hydrolysis of the native VIP molecule. The biological consequences of the binding of VIP and GRF to these macromolecular "receptors" are considered.

Structure-activity studies of vasoactive intestinal peptide (VIP): cyclic disulfide analogs.

Analogs of vasoactive intestinal peptide with cysteine residues incorporated at selected sites within the sequence were prepared by solid phase methods, oxidized to the corresponding cyclic disulfides and purified to homogeneity by preparative HPLC. The cyclic compounds were assayed as smooth muscle relaxants on isolated guinea pig trachea, as bronchodilators in vivo in guinea pigs, and for binding to VIP receptors in guinea pig lung membranes. Of the analogs prepared at the N-terminus, one compound, Ac-[D-Cys6,D-Cys11,Lys12,Nle17,Val26,Th r28]-VIP, was found to be a full agonist with slightly more than one tenth the potency of native VIP. Most other cyclic analogs in the N-terminal region were found to be inactive. A second analog, Ac-[Lys12,Cys17,Val26,Cys28]-VIP, was also found to be a full agonist with potency about one third that of native VIP. Furthermore, this compound was active as a bronchodilator in vivo in guinea pig, but with somewhat diminished potency as compared to native VIP. Strikingly, this cyclic compound was found to have significantly longer duration of action (> 40 min) when compared to an analogous acyclic compound (5 min). The conformational restrictions imposed by formation of the cyclic ring structures may have stabilized the molecule to degradation, thus enhancing the effective duration of action. Analysis of this series of cyclic analogs has also yielded information about the requirements for the receptor-active conformation of VIP.

Interleukin-1 beta induces cytosolic PLA2 in parallel with prostaglandin E2 in rheumatoid synovial fibroblasts.

We investigated the temporal relationship between the increase in enzymatic activity and protein of a high molecular weight (100 kDa), cytosolic PLA2 (cPLA2) in interleukin-1 beta (IL-1 beta)-treated rheumatoid synovial fibroblasts (RSF). Both of these responses increased according to a similar time-course which correlates with PGE2 production by these cells. In contrast, 14 kDa, secreted PLA2 (sPLA2), which was also produced by RSF, was not affected by IL-1 beta treatment. These findings support that an augmentation of CPLA2 activity, caused by an induction of cPLA2 protein, rather than sPLA2, is temporally associated with increased PGE2 production in IL-1 beta-treated RSF.

Antiflammin-2 (HDMNKVLDL) does not inhibit phospholipase A2 activities.

A basic nonapeptide P2 (antiflammin-2, HDMNKVLDL) which is identical to a portion of the amino acid sequence (residues 246-254) of lipocortin I, has been described to have antiinflammatory activity in a rat paw edema model (Nature 335: 726-730 [1988]). P2 (0.05 microM) was also reported to inhibit porcine pancreatic phospholipase A2 (PLA2). The effect of synthetic P2 (98% pure) on PLA2 was evaluated in two assay systems. Using porcine pancreatic PLA2 and phosphatidylcholine/deoxycholate mixed micellar substrate, P2 (0.005-50 microM) had no effect on PLA2 activity, even in the presence of 2-mercaptoethanol to prevent peptide oxidation. In another assay, using human synovial fluid PLA2 as the enzyme and [14C]-oleate-labelled E. coli substrate, P2 (0.005-50 microM) had no significant effect on PLA2 activity. A reported PLA2 inhibitor, manoalide, was a potent inhibitor of PLA2 in both assay systems. On the basis of these results, we conclude that P2 is devoid of PLA2 inhibitory activity.

Structure-activity studies of vasoactive intestinal polypeptide.

This report explores the potential side-chain functional groups required for interaction of the bronchodilator neuropeptide, vasoactive intestinal peptide (VIP), with its receptor. The binding affinity and biological activity of native VIP have been found to be sensitive to the removal of amino- and carboxyl-terminal residues. This data suggests that elements within the entire primary sequence of the VIP molecule appear to be necessary for recognition by VIP receptors. The introduction of alanine residues substituted into the VIP molecule is utilized to probe for side-chain functional groups that are crucial for eliciting high receptor binding affinity in vitro and high biological potency in vivo. The VIP pharmacophore appears to be identical in guinea pig lung and human lung and consists of multiple binding sites most likely involving positions Asp3, Phe6, Thr7, Tyr10, Tyr22, and Leu23. These findings could be exploited to enhance the biological potency of VIP by increasing the binding energy at these positions.